Research Area: Human Disease

Sharpening the edges of cancer chemotherapy
Nicole Davis | Whitehead Institute
July 11, 2018

Cambridge, MA — Tackling unsolved problems is a cornerstone of scientific research, propelled by the power and promise of new technologies. Indeed, one of the shiniest tools in the biomedical toolkit these days is the genome editing system known as CRISPR/Cas9. Whitehead Institute Member David Sabatini and his colleagues pioneered the use of this tool as a foundation for large-scale genetic screens in human cells, turning up a treasure trove of new insights into cellular metabolism, in both normal cells and cancer cells.

When Naama Kanarek, a postdoc in Sabatini’s laboratory, pondered how to apply these state-of-the-art CRISPR/Cas9 screens to her own research, her thoughts turned to a classic cancer chemotherapy drug, methotrexate, which has been in clinical use for nearly seven decades. Often used to treat a form of pediatric leukemia, known as acute lymphoblastic leukemia (ALL), the drug, when deployed as part of a multifaceted treatment plan, can be highly effective. But its power comes at a cost. Because methotrexate can damage not only cancer cells but also healthy tissues, it must be administered with great care. For children who receive high doses of the drug, a mainstay of ALL treatment, that can mean several days spent in the hospital with rigorous clinical monitoring.

In other forms of cancer, methotrexate’s efficacy is more uncertain. For example, in pediatric osteosarcoma, only 65 percent of patients respond. Unfortunately, there is currently no way for doctors to pinpoint who will and who will not.

“From a scientific standpoint, methotrexate is quite special because it was the first metabolic drug to be developed, but much of its biology remains to be discovered — particularly what drives these different responses in patients,” Kanarek says. “So, this is really one of these old, classic questions that has been lingering in the field for some time. We thought we could learn something new.”

And they did. In the July 11 online issue of the journal Nature, Kanarek, Sabatini, and their colleagues report the findings of a CRISPR/Cas9 screen for factors involved in methotrexate sensitivity. The team’s work yielded a surprising set of discoveries that point to the breakdown of histidine — one of several amino acids used by the body to construct proteins — as a critical gatekeeper of cancer cells’ vulnerability to methotrexate. The researchers’ findings not only help illuminate the biology of a well-known cancer chemotherapy, but also suggest a simple dietary supplement that could help broaden its therapeutic window and reduce its toxicity.

 “This study is an example of the power of modern genomic tools to shine a bright light on longstanding questions in human biology,” says senior author David Sabatini, a Member of Whitehead Institute, a professor of biology at Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute (HHMI). “While cancer chemotherapies can be quite effective, their biological effects are often poorly understood. By laying bare their biology, we may be able to devise ways to utilize them more wisely.”

ATTACK THE CANCER, NOT THE PATIENT

The history of methotrexate stretches back to the 1940s, a time when strikingly little was known about the origins of cancer much less how best to treat it. The birth of methotrexate as a chemotherapeutic agent was sparked by the astute observations of Sidney Farber, a pediatric pathologist at Boston Children’s Hospital who cared for children with a variety of maladies, including ALL. In the course of caring for patients with ALL, Farber recognized that cancer cells depended on the nutrient folic acid for their own proliferation. That gave him the idea of using folate antagonists to treat ALL. Methotrexate was developed in 1949 precisely for this purpose and was subsequently shown to induce remission in children with ALL. Fast forward to today, and the drug has evolved into a significant tool in oncologists’ toolkit.

“Methotrexate is a major part of the backbone of chemotherapy treatment across many human cancers,” says Loren Walensky, a pediatric hematologist/oncologist at the Dana-Farber Cancer Institute who is not a study co-author but served as an early adviser on the project and will also play a deeper role in planning future follow-up studies. “It is also used outside of the cancer field for the treatment of several autoimmune diseases.”

He added, “But as with all chemotherapy, the critical issue is how to best use it to inflict maximal damage on the cancer without irreparably harming the patient.”

Kanarek explains how new genetic tools are allowing insights into the sensitivity of cancer cells to methotrexate.

The basic mechanics of methotrexate are fairly well known. The drug inhibits dihydrofolate reductase (DHFR), an enzyme that generates the functional form of folate, known as tetrahydrofolate (THF). THF is essential for preparing the raw materials needed to make nucleic acids, such as DNA, which carries cells’ genetic information, and RNA, a close chemical relative involved in making proteins. “Proliferating cells must duplicate their DNA, so they need a lot of THF,” Kanarek explains. “But even cells that are not dividing need to make RNA, and that requires THF, too.”

The results of Kanarek’s CRISPR/Cas9 screen now bring greater clarity to this molecular picture. She and her colleagues uncovered another enzyme, called FTCD, which is involved in the breakdown of histidine. Interestingly, FTCD also requires THF for its function — though not nearly as much as the main target of methotrexate, DHFR. Despite the differential demands of the two enzymes, they both draw from the same, shared pool of THF.

“Under normal conditions, this pool is sufficiently full, so there is no competition for resources, even in rapidly dividing cells,” Kanarek says.

But when the amount of THF becomes limiting — as it does in cells that are treated with methotrexate — the story is quite different, the Whitehead Institute team discovered. In that case, the activity of FTCD poses serious problems, because there isn’t enough THF in the pool to support both cell proliferation and histidine breakdown. When that happens, the cells die.

That got Kanarek thinking more about histidine: Could the nutrient provide a way to tinker with FTCD activity and, by virtue of the cancer cells’ own metabolism, make them more vulnerable to methotrexate?

To explore this question, the researchers used mouse models of leukemia, engineered by transplanting human leukemia cells under the skin of immunocompromised mice. A subset of the mice received injections of methotrexate together with histidine. This one-two punch, Kanarek hypothesized, should ramp up the function of FTCD and more rapidly drain the THF pool, thereby making the cells more sensitive to the cancer-killing effects of methotrexate.

That is precisely what the team observed. Notably, these experiments involved lower than normal doses of methotrexate, suggesting the cells had indeed been made more sensitive to the cancer drug. Moreover, the studies included a human leukemia cell line, called SEM, which harbors a specific genetic mutation that is associated with a particularly poor prognosis in patients — further underscoring the power of the histidine degradation pathway to weaken cells’ defenses.

Now, Kanarek and her colleagues are working to extend these initial findings with additional preclinical studies and, together with Walensky, determine how to best evaluate the potential benefits of histidine supplementation in cancer patients. Their ultimate goal: to pursue clinical trials that will assess histidine’s ability to improve the effectiveness of methotrexate in humans.

In addition to making cancer cells more vulnerable to methotrexate, the Whitehead Institute team’s research also holds promise for another therapeutic challenge: identifying which patients will or will not respond to the drug.

Two other enzymes cooperate with FTCD in breaking down histidine. The levels of one of the enzymes, known as HAL, appears to correlate with cells’ sensitivity to methotrexate: That is, cancer cells with high levels of HAL tend to be more sensitive to the drug. More work is needed to determine whether this correlation extends to a broader swath of patient samples and if it has predictive value in the clinic. Nevertheless, Kanarek and her colleagues are already beginning work on this front. Together with Abner Louissaint, Jr., a hematopathologist at Massachusetts General Hospital who also served as an early adviser on the Nature study, the Whitehead Institute team will launch a second clinical study to examine whether HAL levels can predict methotrexate response in patients with lymphoma.

“Being able to understand who is going to respond to methotrexate and who is not, and how to achieve a therapeutic benefit while mitigating the drug’s potential side effects, could have a profound impact on patient care,” Walensky says. “The insights from this study bring an entirely new dimension to our understanding of a decades-old and critically important cancer medicine. And as a physician and a scientist, that’s truly exciting.”

Written by Nicole Davis

* * *

David Sabatini’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a Howard Hughes Medical Institute investigator and a professor of biology at Massachusetts Institute of Technology.

* * *

Full citation:

“Histidine catabolism is a major determinant of methotrexate sensitivity”

Nature, online on July 11, 2018.

Naama Kanarek (1,2,3,4), Heather R. Keys (1), Jason R. Cantor (1,2,3,4), Caroline A. Lewis (1), Sze Ham Chan (1), Tenzin Kunchok (1), Monther Abu-Remaileh (1,2,3,4), Elizaveta Freinkman (1), Lawrence D. Schweitzer (4), and David M. Sabatini (1,2,3,4).

  1. Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, 455 main Street, Cambridge, Massachusetts 02142, USA
  2. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
  3. Koch Institute for Integrative Cancer Research and Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA
  4. Broad Institute of Harvard and Massachusetts Institute of Technology, 415 main Street, Cambridge, Massachusetts 02142, USA
Restricting a key cellular nutrient could slow tumor growth

Researchers identify the amino acid aspartate as a metabolic limitation in certain cancers.

Raleigh McElvery | Department of Biology
June 29, 2018

Remove tumor cells from a living organism and place them in a dish, and they will multiply even faster than before. The mystery of why this is has long stumped cancer researchers, though many have simply focused on the mutations and chains of molecular reactions that could prompt such a disparity. Now, a group of MIT researchers suggests that the growth limitations in live organisms may stem from a different source: the cell’s environment. More specifically, they found that the amino acid aspartate serves as a key nutrient needed for the “proliferation” or rapid duplication of cancer cells when oxygen is not freely available.

The biologists took cancer cells from various tissue types and engineered them to convert another, more abundant substrate into aspartate using the gene encoding an enzyme from guinea pigs. This had no effect on the cells sitting in a dish, but the same cells implanted into mice engendered tumors that grew faster than ever before. The researchers had increased the cells’ aspartate supply, and in doing so successfully sped up proliferation in a living entity.

“There hasn’t been a lot of thought into what slows tumor growth in terms of the cellular environment, including the sort of food cancer cells need,” says Matthew Vander Heiden, associate professor of biology, associate director of the Koch Institute for Integrative Cancer Research, and senior author of the study. “For instance, if you’re trying to get to a given destination and I want to slow you down, my best bet is to set up a roadblock at a place on your route where you’d experience a slow-down anyways, like a long traffic light. That’s essentially what we’re interested in here — understanding what nutrients the cell is already lacking that put the brakes on proliferation, and then further limiting those nutrients to inhibit growth even more.”

Lucas Sullivan, a postdoc in Vander Heiden’s lab, is the lead author of the study, which appeared in Nature Cell Biology on June 25.

Building the case for aspartate

Isolating a single factor that could impact tumor growth within an organism is tricky business. One potential candidate came to Sullivan via a paper he co-authored with graduate student Dan Gui in 2015, which asked a somewhat controversial question: Why is it that cells need to consume oxygen through cellular respiration in order to proliferate?

It’s a rather counter-intuitive question, because some scientific literature suggests just the opposite: Cancer cells in an organism (“in vivo”) do not enjoy the same access to oxygen as they would in a dish, and therefore don’t depend on oxygen to produce enough energy to divide. Instead, they switch to a different process, fermentation, that doesn’t require oxygen. But Sullivan and Gui noted that cancer cells do rely on oxygen for another reason: to produce aspartate as a byproduct.

Aspartate, they soon confirmed, does, in fact, play a crucial role in controlling the rate of cancer cell proliferation. In another study one year later, Sullivan and Gui noted that the antidiabetic drug metformin, known to inhibit mitochondria, slowed tumor growth and decreased aspartate levels in cells in vivo. Since mitochondria are key to cellular respiration, Sullivan reasoned that blocking their function in an already oxygen-constrained environment (the tumor) might make cancer cells vulnerable to further suppression of respiration — and aspartate — explaining why metformin seems to have such a strong effect on tumor growth.

Despite being potentially required for certain amino acids and the synthesis of all four DNA nucleotides, aspartate is already hard to come by, even in oxygen-rich environments. It’s among the lowest concentration amino acids in our blood, and has no way to enter our cells unless a rare protein transporter is present. Precisely why aspartate import is so inefficient remains an evolutionary mystery; one possibility is that its scarcity serves as a “failsafe,” preventing cells from multiplying until they have all the resources to properly do so.

Regardless, the easiest way for cells to get aspartate is not to import it from outside, but rather to make it directly inside, breaking down another amino acid called asparagine to generate it. However, there are very few known mammals that have an enzyme capable of producing aspartate from asparagine — among them, the guinea pig.

Channeling the guinea pig

In the 1950s, a researcher named John Kidd made an accidental discovery. He injected cancer-ridden rats with sera from various animals — rabbits, horses, guinea pigs, and the like — and discovered that guinea pig serum alone shrunk the rats’ tumors. It wasn’t until years later that scientists learned it was an enzyme in the guinea pig blood called guinea pig asparaginase 1 (gpASNase1) that was responsible for this antitumorigenic effect. Today, we know about a host of simpler organisms with similar enzymes, including bacteria and zebrafish. In fact, bacterial asparaginase is approved as a medicine to treat acute lymphocytic leukemia.

Because guinea pigs are mammals and thus have similar metabolisms to our own, the MIT researchers decided to use gpASNase1 to increase aspartate levels in tumors in four different tumor types and ask whether the tumors would grow faster. This was the case for three of the four types: The colon cancer cells, osteosarcoma cells, and mouse pancreatic cancer cells divided more rapidly than before, but the human pancreatic cancer cells continued to proliferate at their normal pace.

“This is a relatively small sample, but you could take this to mean that not every cell in the body is as sensitive to loss of aspartate production as others,” Sullivan says. “Acquiring aspartate may be a metabolic limitation for only a subset of cancers, since aspartate can be produced via a number of different pathways, not just through asparagine conversion.”

When the researchers tried to slow tumor growth using the antidiabetic metformin, the cells expressing gpASNase1 remained unaffected — confirming Sullivan’s prior suspicion that metformin slows tumor growth specifically by impeding cellular respiration and suppressing aspartate production.

“Our initial finding connecting metformin and proliferation was very serendipitous,” he says, “but these most recent results are a clear proof of concept. They show that decreasing aspartate levels also decreases tumor growth, at least in some tumors. The next step is to determine if there are other ways to more intentionally target aspartate synthesis in certain tissues and improve our current therapeutic approaches.”

Although the efficacy of using metformin to treat cancer remains controversial, these findings indicate that one means to target tumors would be to prevent them from accessing or producing nutrients like aspartate to make new cells.

“Although there are many limitations to cancer cell proliferation, which metabolites become limiting for tumor growth has been poorly understood,” says Kivanc Birsoy, the Chapman-Perelman Assistant Professor at Rockefeller University. “This study identifies aspartate as one such limiting metabolite, and suggests that its availability could be targeted for anti-cancer therapies.”

Birsoy is a former postdoc in professor of biology David Sabatini’s lab, who authored a paper published in the same issue of Nature Cell Biology, identifying aspartate as a major growth limitation in oxygen-deprived tumors.

“These companion papers demonstrate that some tumors in vivo are really limited by the chemical processes that require oxygen to get the aspartate they need to grow, which can affect their sensitivity to drugs like metformin,” Vander Heiden says. “We’re beginning to realize that understanding which cancer patients will respond to which treatments may be determined by factors besides genetic mutations. To really get the full picture, we need to take into account where the tumor is located, its nutrient availability, and the environment in which it lives.”

The research was funded by an NIH Pathway to Independence Award, the American Cancer Society, Ludwig Center for Molecular Oncology Fund, the National Science Foundation, a National Institutes of Health Ruth Kirschstein Fellowship, Alex’s Lemonade Stand Undergraduate Research Fellowship, Damon Runyon Cancer Research Foundation, Howard Hughes Medical Institute Faculty Scholar Award, Stand Up to Cancer, Lustgarten Foundation, Ludwig Center at MIT, the National Institutes of Health, and the Koch Institute’s Center for Precision Cancer Medicine.

Stem cell-derived zika model suggests mechanisms underlying microcephaly
Nicole Giese Rura | Whitehead Institute
June 21, 2018

Cambridge, MA  – Scientists turn to model organisms, like mice and yeast, to investigate the biology underlying emerging diseases. But for the Zika virus, the lack of a good model hampered this type of research. Now, a team of researchers in the laboratory of Whitehead Institute Founding Member Rudolf Jaenisch has devised a way to model Zika and other neural diseases in a dish. Their work is described this week in the journal PNAS.

The Zika virus was identified in 1947 in Uganda, but a 2013 epidemic in French Guinea first brought it to the public’s attention. As the disease spread throughout the Americas and the Caribbean in 2014, abnormalities, such as microcephaly in newborns, were increasingly reported when mothers were infected during their first trimester. Scientists’ efforts to better understand the virus and its mechanisms quickly hit a snag: mice, which are often used to model disease pathology, are not vulnerable to the Zika virus unless their innate immune defenses are knocked out. Additionally, neural diseases, such as those that cause microcephaly, affect cells that reside deep in the brain, and they cannot be easily accessed for observation and manipulation.

In order to circumvent these challenges and to model Zika in the lab, the researchers turned to induced pluripotent stem cells (iPSCs)–adult cells that have been pushed back to a embryonic stem cell-like state. iPSCs can in turn be nudged to mature into almost any cell type in the body. In previous work, Julien Muffat and Yun Li, former postdoctoral researchers in the Jaenisch lab, were the first to use iPSCs to create microglia, the specialized immune cells that maintain the brain and spinal cord and care for them after injury.

In the current work, Muffat and Li teamed up with Attya Omer, also a graduate student in the Jaenisch lab, and Lee Gehrke’s lab at MIT to study the effect of the Zika virus on iPSC-derived versions of three neural cell types critical during human fetal brain development: microglia, neural progenitors, and astrocytes. Whether the Zika virus can infect these cells and how well the cells can clear the virus could provide insight into why the virus can cause birth defects like microcephaly. Using their model, the team determined that after being infected with a strain derived from the initial Ugandan Zika virus, microglia can survive and can continue to harbor the virus. This is important because in a developing embryo, microglia move from the yolk sac to the developing brain very early in gestation. The study shows that, like their in vivo counterparts, iPSC-derived microglia could invade the immature neural tissue of a brain organoid, and pre-infected microglia could transfer the virus to the organoids. According to Muffat, this suggests that if microglial precursors are infected before their journey, they could shuttle the Zika virus to the developing brain and infect the neural progenitors residing there.

Neural progenitor cells, which during gestation produce the neurons and glia that constitute the majority of the human brain, are particularly vulnerable to the Zika virus and die when infected. To better understand why these cells are so susceptible, the team compared how the Zika virus and the closely related dengue virus affect the neural progenitor cells. Dengue, which does not cause birth defects like microcephaly, triggers a strong cellular immune response, called interferon, in the neural progenitors, which enables the progenitor cells to efficiently fight and clear the dengue virus. In sharp contrast, when exposed to the Zika virus, neural progenitors mount little if any interferon immune defense. Pretreating the neural progenitor cells with interferon before exposure to the Zika virus impedes the virus’s progression and proliferation, and reduces cell death. These results suggest that therapeutically altering interferon levels could prevent some of the more dire effects of Zika infection on the neural progenitor cells.

According to the team, using iPSC-derived cells has great potential for modeling Zika virus as well as many other diseases that affect the central nervous system.

This work was supported by the European Leukodystrophy Association, the Brain & Behavior Research Foundation, the Simons Foundation (SFARI 204106), the International Rett Syndrome Foundation, Howard Hughes Medical Institute, the National Institutes of Health (NIH grants HD 045022, R37-CA084198, AI100190), the ELA Foundation, the Emerald Foundation, and Biogen. Jaenisch is a cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

Written by Nicole Giese Rura
***
Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.
 ***
Full citation:
“Human Induced Pluripotent Stem Cell-derived Glial Cells and Neural Progenitors Display Divergent Responses to Zika and Dengue Infections”
PNAS, online June 18, 2018.
Julien Muffat (1,8), Yun Li (1,8), Attya Omer (1,8), Ann Durbin (3,4,5), Irene Bosch (3,4,5), Grisilda Bakiasi (6), Edward Richards (7), Aaron Meyer (7), Lee Gehrke (3,4,5), Rudolf Jaenisch (1,2).
1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, MA 02139, USA
3. IMES, Massachusetts Institute of Technology, Cambridge MA 02139, USA
4. Department of Microbiology and Immunobiology, Harvard Medical School, Boston 02115, USA
5. Harvard-MIT Program in Health Sciences and Technology, Cambridge, MA 02139, USA
6. Bryn Mawr College, Bryn Mawr, PA
7. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA
8. These authors contributed equally
Biologists discover how pancreatic tumors lead to weight loss

Shortfall of digestive enzymes can lead to tissue breakdown in early stages of pancreatic cancer.

Anne Trafton | MIT News Office
June 20, 2018

Patients with pancreatic cancer usually experience significant weight loss, which can begin very early in the disease. A new study from MIT and Dana-Farber Cancer Institute offers insight into how this happens, and suggests that the weight loss may not necessarily affect patients’ survival.

In a study of mice, the researchers found that weight loss occurs due to a reduction in key pancreatic enzymes that normally help digest food. When the researchers treated these mice with replacement enzymes, they were surprised to find that while the mice did regain weight, they did not survive any longer than untreated mice.

Pancreatic cancer patients are sometimes given replacement enzymes to help them gain weight, but the new findings suggest that more study is needed to determine whether that actually benefits patients, says Matt Vander Heiden, an associate professor of biology at MIT and a member of the Koch Institute for Integrative Cancer Research.

“We have to be very careful not to draw medical advice from a mouse study and apply it to humans,” Vander Heiden says. “The study does raise the question of whether enzyme replacement is good or bad for patients, which needs to be studied in a clinical trial.”

Vander Heiden and Brian Wolpin, an associate professor of medicine at Harvard Medical School and Dana-Farber Cancer Institute, are the senior authors of the study, which appears in the June 20 issue of Nature. The paper’s lead authors are Laura Danai, a former MIT postdoc, and Ana Babic, an instructor in medicine at Dana-Farber.

Starvation mode

In a 2014 study, Vander Heiden and his colleagues found that muscle starts breaking down very early in pancreatic cancer patients, usually long before any other signs of the disease appear.

Still unknown was how this tissue wasting process occurs. One hypothesis was that pancreatic tumors overproduce some kind of signaling factor, such as a hormone, that circulates in the bloodstream and promotes breakdown of muscle and fat.

However, in their new study, the MIT and Dana-Farber researchers found that this was not the case. Instead, they discovered that even very tiny, early-stage pancreatic tumors can impair the production of key digestive enzymes. Mice with these early-stage tumors lost weight even though they ate the same amount of food as normal mice. These mice were unable to digest all of their food, so they went into a starvation mode where the body begins to break down other tissues, especially fat.

The researchers found that when they implanted pancreatic tumor cells elsewhere in the body, this weight loss did not occur. That suggests the tumor cells are not secreting a weight-loss factor that circulates in the bloodstream; instead, they only stimulate tissue wasting when they are in the pancreas.

The researchers then explored whether reversing this weight loss would improve survival. Treating the mice with pancreatic enzymes did reverse the weight loss. However, these mice actually survived for a shorter period of time than mice that had pancreatic tumors but did not receive the enzymes. That finding, while surprising, is consistent with studies in mice that have shown that calorie restriction can have a protective effect against cancer and other diseases.

“It turns out that this mechanism of tissue wasting is actually protective, at least for the mice, in the same way that limiting calories can be protective for mice,” Vander Heiden says.

Human connection

The intriguing findings from the mouse study prompted the research team to see if they could find any connection between weight loss and survival in human patients. In an analysis of medical records and blood samples from 782 patients, they found no link between degree of tissue wasting at the time of diagnosis and length of survival. That finding is important because it could reassure patients that weight loss does not necessarily mean that the patient will do worse, Vander Heiden says.

“Sometimes you can’t do anything about this weight loss, and this finding may mean that just because the patient is eating less and is losing weight, that doesn’t necessarily mean that they’re shortening their life,” he says.

The researchers say that more study is needed to determine if the same mechanism they discovered in mice is also occurring in human cancer patients. Because the mechanism they found is very specific to pancreatic tumors, it may differ from the underlying causes behind tissue wasting seen in other types of cancer and diseases such as HIV.

“From a mechanistic standpoint, this study reveals a very different way to think about what could be causing at least some weight loss in pancreatic cancer, suggesting that not all weight loss is the same across different cancers,” Vander Heiden says. “And it raises questions that we really need to study more, because some mechanisms may be protective and some mechanisms may be bad for you.”

Clary Clish, director of the Metabolomics Platform at the Broad Institute, and members of his research group also contributed to this work. The research was funded, in part, by the Lustgarten Foundation, a National Institutes of Health Ruth Kirschstein Fellowship, Stand Up 2 Cancer, the Ludwig Center for Molecular Oncology at MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the MIT Center for Precision Cancer Medicine, and the National Institutes of Health.

Biologists discover function of gene linked to familial ALS

Study in worms reveals gene loss can lead to accumulation of waste products in cells.

Anne Trafton | MIT News Office
May 4, 2018

MIT biologists have discovered a function of a gene that is believed to account for up to 40 percent of all familial cases of amyotrophic lateral sclerosis (ALS). Studies of ALS patients have shown that an abnormally expanded region of DNA in a specific region of this gene can cause the disease.

In a study of the microscopic worm Caenorhabditis elegans, the researchers found that the gene has a key role in helping cells to remove waste products via structures known as lysosomes. When the gene is mutated, these unwanted substances build up inside cells. The researchers believe that if this also happens in neurons of human ALS patients, it could account for some of those patients’ symptoms.

“Our studies indicate what happens when the activities of such a gene are inhibited — defects in lysosomal function. Certain features of ALS are consistent with their being caused by defects in lysosomal function, such as inflammation,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, and the senior author of the study.

Mutations in this gene, known as C9orf72, have also been linked to another neurodegenerative brain disorder known as frontotemporal dementia (FTD), which is estimated to affect about 60,000 people in the United States.

“ALS and FTD are now thought to be aspects of the same disease, with different presentations. There are genes that when mutated cause only ALS, and others that cause only FTD, but there are a number of other genes in which mutations can cause either ALS or FTD or a mixture of the two,” says Anna Corrionero, an MIT postdoc and the lead author of the paper, which appears in the May 3 issue of the journal Current Biology.

Genetic link

Scientists have identified dozens of genes linked to familial ALS, which occurs when two or more family members suffer from the disease. Doctors believe that genetics may also be a factor in nonfamilial cases of the disease, which are much more common, accounting for 90 percent of cases.

Of all ALS-linked mutations identified so far, the C9orf72 mutation is the most prevalent, and it is also found in about 25 percent of frontotemporal dementia patients. The MIT team set out to study the gene’s function in C. elegans, which has an equivalent gene known as alfa-1.

In studies of worms that lack alfa-1, the researchers discovered that defects became apparent early in embryonic development. C. elegans embryos have a yolk that helps to sustain them before they hatch, and in embryos missing alfa-1, the researchers found “blobs” of yolk floating in the fluid surrounding the embryos.

This led the researchers to discover that the gene mutation was affecting the lysosomal degradation of yolk once it is absorbed into the cells. Lysosomes, which also remove cellular waste products, are cell structures which carry enzymes that can break down many kinds of molecules.

When lysosomes degrade their contents — such as yolk — they are reformed into tubular structures that split, after which they are able to degrade other materials. The MIT team found that in cells with the alfa-1 mutation and impaired lysosomal degradation, lysosomes were unable to reform and could not be used again, disrupting the cell’s waste removal process.

“It seems that lysosomes do not reform as they should, and material accumulates in the cells,” Corrionero says.

For C. elegans embryos, that meant that they could not properly absorb the nutrients found in yolk, which made it harder for them to survive under starvation conditions. The embryos that did survive appeared to be normal, the researchers say.

Neuronal effects

The researchers were able to partially reverse the effects of alfa-1 loss in the C. elegans embryos by expressing the human protein encoded by the c9orf72 gene. “This suggests that the worm and human proteins are performing the same molecular function,” Corrionero says.

If loss of C9orf72 affects lysosome function in human neurons, it could lead to a slow, gradual buildup of waste products in those cells. ALS usually affects cells of the motor cortex, which controls movement, and motor neurons in the spinal cord, while frontotemporal dementia affects the frontal areas of the brain’s cortex.

“If you cannot degrade things properly in cells that live for very long periods of time, like neurons, that might well affect the survival of the cells and lead to disease,” Corrionero says.

Many pharmaceutical companies are now researching drugs that would block the expression of the mutant C9orf72. The new study suggests certain possible side effects to watch for in studies of such drugs.

“If you generate drugs that decrease c9orf72 expression, you might cause problems in lysosomal homeostasis,” Corrionero says. “In developing any drug, you have to be careful to watch for possible side effects. Our observations suggest some things to look for in studying drugs that inhibit C9orf72 in ALS/FTD patients.”

The research was funded by an EMBO postdoctoral fellowship, an ALS Therapy Alliance grant, a gift from Rose and Douglas Barnard ’79 to the McGovern Institute, and a gift from the Halis Family Foundation to the MIT Aging Brain Initiative.

Fasting boosts stem cells’ regenerative capacity

A drug treatment that mimics fasting can also provide the same benefit, study finds.

Anne Trafton | MIT News Office
May 1, 2018

As people age, their intestinal stem cells begin to lose their ability to regenerate. These stem cells are the source for all new intestinal cells, so this decline can make it more difficult to recover from gastrointestinal infections or other conditions that affect the intestine.

This age-related loss of stem cell function can be reversed by a 24-hour fast, according to a new study from MIT biologists. The researchers found that fasting dramatically improves stem cells’ ability to regenerate, in both aged and young mice.

In fasting mice, cells begin breaking down fatty acids instead of glucose, a change that stimulates the stem cells to become more regenerative. The researchers found that they could also boost regeneration with a molecule that activates the same metabolic switch. Such an intervention could potentially help older people recovering from GI infections or cancer patients undergoing chemotherapy, the researchers say.

“Fasting has many effects in the intestine, which include boosting regeneration as well as potential uses in any type of ailment that impinges on the intestine, such as infections or cancers,” says Omer Yilmaz, an MIT assistant professor of biology, a member of the Koch Institute for Integrative Cancer Research, and one of the senior authors of the study. “Understanding how fasting improves overall health, including the role of adult stem cells in intestinal regeneration, in repair, and in aging, is a fundamental interest of my laboratory.”

David Sabatini, an MIT professor of biology and member of the Whitehead Institute for Biomedical Research and the Koch Institute, is also a senior author of the paper, which appears in the May 3 issue of Cell Stem Cell.

“This study provided evidence that fasting induces a metabolic switch in the intestinal stem cells, from utilizing carbohydrates to burning fat,” Sabatini says. “Interestingly, switching these cells to fatty acid oxidation enhanced their function significantly. Pharmacological targeting of this pathway may provide a therapeutic opportunity to improve tissue homeostasis in age-associated pathologies.”

The paper’s lead authors are Whitehead Institute postdoc Maria Mihaylova and Koch Institute postdoc Chia-Wei Cheng.

Boosting regeneration

For many decades, scientists have known that low caloric intake is linked with enhanced longevity in humans and other organisms. Yilmaz and his colleagues were interested in exploring how fasting exerts its effects at the molecular level, specifically in the intestine.

Intestinal stem cells are responsible for maintaining the lining of the intestine, which typically renews itself every five days. When an injury or infection occurs, stem cells are key to repairing any damage. As people age, the regenerative abilities of these intestinal stem cells decline, so it takes longer for the intestine to recover.

“Intestinal stem cells are the workhorses of the intestine that give rise to more stem cells and to all of the various differentiated cell types of the intestine. Notably, during aging, intestinal stem function declines, which impairs the ability of the intestine to repair itself after damage,” Yilmaz says. “In this line of investigation, we focused on understanding how a 24-hour fast enhances the function of young and old intestinal stem cells.”

After mice fasted for 24 hours, the researchers removed intestinal stem cells and grew them in a culture dish, allowing them to determine whether the cells can give rise to “mini-intestines” known as organoids.

The researchers found that stem cells from the fasting mice doubled their regenerative capacity.

“It was very obvious that fasting had this really immense effect on the ability of intestinal crypts to form more organoids, which is stem-cell-driven,” Mihaylova says. “This was something that we saw in both the young mice and the aged mice, and we really wanted to understand the molecular mechanisms driving this.”

Metabolic switch

Further studies, including sequencing the messenger RNA of stem cells from the mice that fasted, revealed that fasting induces cells to switch from their usual metabolism, which burns carbohydrates such as sugars, to metabolizing fatty acids. This switch occurs through the activation of transcription factors called PPARs, which turn on many genes that are involved in metabolizing fatty acids.

The researchers found that if they turned off this pathway, fasting could no longer boost regeneration. They now plan to study how this metabolic switch provokes stem cells to enhance their regenerative abilities.

They also found that they could reproduce the beneficial effects of fasting by treating mice with a molecule that mimics the effects of PPARs. “That was also very surprising,” Cheng says. “Just activating one metabolic pathway is sufficient to reverse certain age phenotypes.”

Jared Rutter, a professor of biochemistry at the University of Utah School of Medicine, described the findings as “interesting and important.”

“This paper shows that fasting causes a metabolic change in the stem cells that reside in this organ and thereby changes their behavior to promote more cell division. In a beautiful set of experiments, the authors subvert the system by causing those metabolic changes without fasting and see similar effects,” says Rutter, who was not involved in the research. “This work fits into a rapidly growing field that is demonstrating that nutrition and metabolism has profound effects on the behavior of cells and this can predispose for human disease.”

The findings suggest that drug treatment could stimulate regeneration without requiring patients to fast, which is difficult for most people. One group that could benefit from such treatment is cancer patients who are receiving chemotherapy, which often harms intestinal cells. It could also benefit older people who experience intestinal infections or other gastrointestinal disorders that can damage the lining of the intestine.

The researchers plan to explore the potential effectiveness of such treatments, and they also hope to study whether fasting affects regenerative abilities in stem cells in other types of tissue.

The research was funded by the National Institutes of Health, the V Foundation, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the Kathy and Curt Marble Cancer Research Fund, the MIT Stem Cell Initiative through Fondation MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, the Damon Runyon Cancer Research Foundation, the Robert Black Charitable Foundation, a Koch Institute Ludwig Postdoctoral Fellowship, a Glenn/AFAR Breakthroughs in Gerontology Award, and the Howard Hughes Medical Institute.

Structure of key growth regulator revealed

Researchers identify the molecular structure of the GATOR1 protein complex, which regulates growth signals in human cells, using cryo-electron microscopy.

Nicole Davis | Whitehead Institute
March 28, 2018

A team of researchers from Whitehead Institute and the Howard Hughes Medical Institute has revealed the structure of a key protein complex in humans that transmits signals about nutrient levels, enabling cells to align their growth with the supply of materials needed to support that growth. This complex, called GATOR1, acts as a kind of on-off switch for the “grow” (or “don’t grow”) signals that flow through a critical cellular growth pathway known as mTORC1.

Despite its importance, GATOR1 bears little similarity to known proteins, leaving major gaps in scientists’ understanding of its molecular structure and function. Now, as described online on March 28 in the journal Nature, Whitehead scientists and their colleagues have generated the first detailed molecular picture of GATOR1, revealing a highly ordered group of proteins and an extremely unusual interaction with its partner, the Rag GTPase.

“If you know something about a protein’s three-dimensional structure, then you can make some informed guesses about how it might work. But GATOR1 has basically been a black box,” says senior author David Sabatini, a member of Whitehead Institute, a professor of biology at MIT, and investigator with the Howard Hughes Medical Institute (HHMI). “Now, for the first time, we have generated high-resolution images of GATOR1 and can begin to dissect how this critical protein complex works.”

GATOR1 was first identified about five years ago. It consists of three protein subunits (Depdc5, Nprl2, and Nprl3), and mutations in these subunits have been associated with human diseases, including cancers and neurological conditions such as epilepsy. However, because of the lack of similarity to other proteins, the majority of the GATOR1 complex is a molecular mystery. “GATOR1 has no well-defined protein domains,” explains Whitehead researcher Kuang Shen, one of the study’s first authors. “So, this complex is really quite special and also very challenging to study.”

Because of the complex’s large size and relative flexibility, GATOR1 cannot be readily crystallized — a necessary step for resolving protein structure through standard, X-ray crystallographic methods. As a result, Shen and Sabatini turned to HHMI’s Zhiheng Yu. Yu and his team specialize in cryo-electron microscopy (cryo-EM), an emerging technique that holds promise for visualizing the molecular structures of large proteins and protein complexes. Importantly, it does not utilize protein crystals. Instead, proteins are rapidly frozen in a thin layer of vitrified ice and then imaged by a beam of fast electrons inside an electron microscope column.

“There have been some major advances in cryo-EM technology over the last decade, and now, it is possible to achieve atomic or near atomic resolution for a variety of proteins,” explains Yu, a senior author of the paper and director of HHMI’s shared, state-of-the-art cryo-EM facility at Janelia Research Campus. Last year’s Nobel Prize in chemistry was awarded to three scientists for their pioneering efforts to develop cryo-EM.

GATOR1 proved to be a tricky subject, even for cryo-EM, and required some trial-and-error on the part of Yu, Shen, and their colleagues to prepare samples that could yield robust structural information. Moreover, the team’s work was made even more difficult by the complex’s unique form. With no inklings of GATOR1’s potential structure, Shen and his colleagues, including co-author Edward Brignole of MIT, had to derive it completely from scratch.

Nevertheless, the Whitehead-HHMI team was able to resolve near-complete structures for GATOR1 as well as for GATOR1 bound to its partner proteins, the Rag GTPases. (Two regions of the subunit Depdc5 are highly flexible and therefore could not be resolved.) From this wealth of new information as well as from the team’s subsequent biochemical analyses, some surprising findings emerged.

First is the remarkable level of organization of GATOR1. The protein is extremely well organized, which is quite unusual for proteins that have no predicted structures. (Such proteins are usually quite disorganized.) In addition, the researchers identified four protein domains that have never before been visualized. These novel motifs — named NTD, SABA, SHEN, and CTD — could provide crucial insights into the inner workings of the GATOR1 complex.

Shen, Sabatini, and their colleagues uncovered another surprise. Unlike other proteins that bind to Rag GTPases, GATOR1 contacts these proteins at at least two distinct sites. Moreover, one of the binding sites serves to inhibit — rather than stimulate — the activity of the Rag GTPase. “This kind of dual binding has never been observed — it is highly unusual,” Shen says. The researchers hypothesize that this feature is one reason why GATOR1 is so large — because it must hold its Rag GTPase at multiple sites, rather than one, as most other proteins of this type do.

Despite these surprises, the researchers acknowledge that their analyses have only begun to scratch the surface of GATOR1 and the mechanisms through which it regulates the mTOR signaling pathway.

“There is much left to discover in this protein,” Sabatini says.

This work was supported by the National Institutes of Health, Department of Defense, National Science Foundation, the Life Sciences Research Foundation, and the Howard Hughes Medical Institute.

Novel human/mouse model could boost type 1 diabetes research
Nicole Giese Rura | Whitehead Institute
March 27, 2018

Cambridge, MA – About 1.5 million people in the United States have type 1 diabetes, according to the Centers for Disease Control and Prevention (CDC), and yet doctors know very little about what triggers the disease. Now researchers at Whitehead Institute have developed a novel platform with human beta cells that could allow scientists to better understand the mechanisms underlying this disease and what provokes it.

In Type 1 diabetes, an autoimmune disease also called juvenile or insulin-dependent diabetes, the immune system destroys beta cells—the cells in the pancreas that produce insulin. Insulin is required for glucose to enter the body’s cells, so people with type 1 diabetes must closely monitor their glucose levels and take insulin daily. Type 1 diabetes is usually diagnosed during childhood or young adulthood, and possible causes of the disease that are being actively researched include genetics, viral infection, other environmental factors, or some combination of these.

Currently, scientists studying the disease may use animal models, such as non-obese diabetic (NOD) mice that do not include human cells, or mouse and rat models with beta cells derived from human induced pluripotent stem cells (iPSCs)—cells that have been pushed to a pluripotent state—implanted into the animals’ kidney capsules. These models hint at clinical applications that may control glucose levels in type 1 diabetes patients, but because the beta cells do not reside in the pancreas, the models do not reflect the cell-tissue interactions that are likely intrinsic in the development of type 1 diabetes.

To address these shortcomings, a team of researchers led by Haiting Ma, a postdoctoral researcher in Whitehead Founding Member Rudolf Jaenisch’s lab, implanted beta cells derived from iPSCs into the pancreas of neonatal mice. As the mice grow, the human beta cells become integrated into the mice’s pancreases, respond to increased glucose levels, and secrete insulin into the mouse’s bloodstream for several months following implantation. The team’s work is described online in the journal PNAS this week.

Using mice with human beta cells successfully engrafted into their pancreases, scientists will be able to study how beta cells function in normal and disease conditions, and perhaps help identify the causes of type 1 diabetes. Such insights may lead to new approaches to treat this autoimmune disease.

This work was supported by Liliana and Hillel Bachrach, the National Institutes of Health (NIH RO1-CA084198, 5R01-MH104610-16, R37-HD045022, R01-GM114864, RF1-AG048029, U19-AI3115135, and 1R01-1NS088538-01), the Harvard Stem Cell Institute, the JBP Foundation, and Howard Hughes Medical Institute. Jaenisch is co-founder advisor of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics, and Doug Melton is the founder of Semma Therapeutics.

* * *
Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.
* * *
Full Citation:
“Establishment of human pluripotent stem cell derived pancreatic β-like cells in the mouse pancreas”
PNAS, online March 26, 2018.
Haiting Ma (1), Katherine Wert (1), Dmitry Shvartsman (2), Douglas Melton (2), and Rudolf Jaenisch (1,3).
1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA
3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
Study suggests method for boosting growth of blood vessels and muscle

Activating proteins linked to longevity may help to increase endurance and combat frailty in the elderly.

Anne Trafton | MIT News Office
March 22, 2018

As we get older, our endurance declines, in part because our blood vessels lose some of their capacity to deliver oxygen and nutrients to muscle tissue. An MIT-led research team has now found that it can reverse this age-related endurance loss in mice by treating them with a compound that promotes new blood vessel growth.

The study found that the compound, which re-activates longevity-linked proteins called sirtuins, promotes the growth of blood vessels and muscle, boosting the endurance of elderly mice by up to 80 percent.

If the findings translate to humans, this restoration of muscle mass could help to combat some of the effects of age-related frailty, which often lead to osteoporosis and other debilitating conditions.

“We’ll have to see if this plays out in people, but you may actually be able to rescue muscle mass in an aging population by this kind of intervention,” says Leonard Guarente, the Novartis Professor of Biology at MIT and one of the senior authors of the study. “There’s a lot of crosstalk between muscle and bone, so losing muscle mass ultimately can lead to loss of bone, osteoporosis, and frailty, which is a major problem in aging.”

The first author of the paper, which appears in Cell on March 22, is Abhirup Das, a former postdoc in Guarente’s lab who is now at the University of New South Wales in Australia. Other senior authors of the paper are David Sinclair, a professor at Harvard Medical School and the University of New South Wales, and Zolt Arany, a professor at the University of Pennsylvania.

Race against time

In the early 1990s, Guarente discovered that sirtuins, a class of proteins found in nearly all animals, protect against the effects of aging in yeast. Since then, similar effects have been seen in many other organisms.

In their latest study, Guarente and his colleagues decided to explore the role of sirtuins in endothelial cells, which line the inside of blood vessels. To do that, they deleted the gene for SIRT1, which encodes the major mammalian sirtuin, in endothelial cells of mice. They found that at 6 months of age, these mice had reduced capillary density and could run only half as far as normal 6-month-old mice.

The researchers then decided to see what would happen if they boosted sirtuin levels in normal mice as they aged. They treated the mice with a compound called NMN, which is a precursor to NAD, a coenzyme that activates SIRT1. NAD levels normally drop as animals age, which is believed to be caused by a combination of reduced NAD production and faster NAD degradation.

After 18-month-old mice were treated with NMN for two months, their capillary density was restored to levels typically seen in young mice, and they experienced a 56 to 80 percent improvement in endurance. Beneficial effects were also seen in mice up to 32 months of age (comparable to humans in their 80s).

“In normal aging, the number of blood vessels goes down, so you lose the capacity to deliver nutrients and oxygen to tissues like muscle, and that contributes to decline,” Guarente says. “The effect of the precursors that boost NAD is to counteract the decline that occurs with normal aging, to reactivate SIRT1, and to restore function in endothelial cells to give rise to more blood vessels.”

These effects were enhanced when the researchers treated the mice with both NMN and hydrogen sulfide, another sirtuin activator.

Vittorio Sartorelli, a principal investigator at the National Institute of Allergy and Infectious Diseases who was not involved in the research, described the experiments as “elegant and compelling.” He added that “it will be of interest and of clinical relevance to evaluate the effect of NMN and hydrogen sulfide on the vascularization of other organs such as the heart and brain, which are often damaged by acutely or chronically reduced blood flow.”

Benefits of exercise

The researchers also found that SIRT1 activity in endothelial cells is critical for the beneficial effects of exercise in young mice. In mice, exercise generally stimulates growth of new blood vessels and boosts muscle mass. However, when the researchers knocked out SIRT1 in endothelial cells of 10-month-old mice, then put them on a four-week treadmill running program, they found that the exercise did not produce the same gains seen in normal 10-month-old mice on the same training plan.

If validated in humans, the findings would suggest that boosting sirtuin levels may help older people retain their muscle mass with exercise, Guarente says. Studies in humans have shown that age-related muscle loss can be partially staved off with exercise, especially weight training.

“What this paper would suggest is that you may actually be able to rescue muscle mass in an aging population by this kind of intervention with an NAD precursor,” Guarente says.

In 2014, Guarente started a company called Elysium Health, which sells a dietary supplement containing a different precursor of NAD, known as NR, as well as a compound called pterostilbene, which is an activator of SIRT1.

The research was funded by the Glenn Foundation for Medical Research, the Sinclair Gift Fund, a gift from Edward Schulak, and the National Institutes of Health.